Low-Molecular-Weight Iron Chelates May Be an Alternative to Gadolinium-based Contrast Agents for T1-weighted Contrast-enhanced MR Imaging.

Purpose To synthesize two low-molecular-weight iron chelates and compare their T1 contrast effects with those of a commercial gadolinium-based contrast agent for their applicability in dynamic contrast material-enhanced (DCE) magnetic resonance (MR) imaging. Materials and Methods The animal experiments were approved by the local ethics committee. Two previously described iron (Fe) chelates of pentetic acid (Fe-DTPA) and of trans-cyclohexane diamine tetraacetic acid (Fe-tCDTA) were synthesized with stability constants several orders of magnitude higher than those of gadolinium-based contrast agents. The T1 contrast effects of the two chelates were compared with those of gadopentetate dimeglumine in blood serum phantoms at 1.5 T, 3 T, and 7 T. For in vivo studies, a human breast cancer cell line (MDA-231) was implanted in five mice per group. The dynamic contrast effects of the chelates were compared by performing DCE MR imaging with intravenous application of Fe-DTPA or Fe-tCDTA on day 1 and DCE MR imaging in the same tumors with gadopentetate dimeglumine on day 2. Quantitative DCE maps were generated with software and were compared by means of a one-tailed Pearson correlation test. Results Relaxivities in serum (0.94 T at room temperature) of Fe-tCDTA (r1 = 2.2 mmol-1 · sec-1, r2 = 2.5 mmol-1 · sec-1) and Fe-DTPA (r1 = 0.9 mmol-1 · sec-1, r2 = 0.9 mmol-1 · sec-1) were approximately twofold and fivefold lower, respectively, compared with those of gadopentetate dimeglumine (r1 = 4.1 mmol-1 · sec-1, r2 = 4.8 mmol-1 · sec-1). Used at moderately higher concentrations, however, iron chelates generated similar contrast effects at T1-weighted MR imaging in vitro in serum, in vivo in blood, and for DCE MR imaging of breast cancer xenografts. The volume transfer constant values for Fe-DTPA and Fe-tCDTA in the same tumors correlated well with those observed for gadopentetate dimeglumine (Fe-tCDTA Pearson R, 0.99; P = .0003; Fe-DTPA Pearson R, 0.97; P = .003). Conclusion Iron-based contrast agents are promising as alternatives for contrast enhancement at T1-weighted MR imaging and have the potential to contribute to the safety of MR imaging. © RSNA, 2017 Online supplemental material is available for this article.

Labeling of mesenchymal stem cells for MRI with single-cell sensitivity.

Sensitive cell detection by magnetic resonance imaging (MRI) is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP) and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP) designed by our department for magnetic particle imaging (MPI) with discontinued Resovist(®) regarding their suitability for detection of single mesenchymal stem cells (MSC) by MRI. We achieved an average intracellular nanoparticle (NP) load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist(®) in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist(®) for improved MRI of MSC with single-cell sensitivity.

Focal liver lesions: SPIO-, gadolinium-, and ferucarbotran-enhanced dynamic T1-weighted and delayed T2-weighted MR imaging in rabbits.

PURPOSE: To compare a superparamagnetic iron oxide (SPIO), VSOP-C184, with a gadopentetate dimeglumine with regard to signal-enhancing effects on T1-weighted dynamic magnetic resonance (MR) images and with another SPIO contrast medium with regard to signal-reducing effects on delayed T2-weighted MR images. MATERIALS AND METHODS: All experiments were approved by the responsible Animal Care Committee. Twenty rabbits (five for each contrast agent and dose) implanted with VX-2 carcinoma were imaged at 1.5 T. VSOP-C184 at 0.015 and 0.025 mmol Fe/kg was compared with gadopentetate dimeglumine at 0.15 mmol Gd/kg and ferucarbotran at 0.015 mmol Fe/kg. The imaging protocol comprised a T1-weighted dynamic gradient-echo (GRE) MR before injection and at 6-second intervals for up to 42 seconds after injection and a T2-weighted turbo spin-echo MR before and 5 minutes after injection. Images were evaluated quantitatively, and contrast media were compared by using nonparametric analysis of variance. RESULTS: At dynamic T1-weighted GRE MR imaging with 0.015-mmol Fe/kg VSOP-C184, 0.025-mmol Fe/kg VSOP-C184, gadopentetate dimeglumine, and ferucarbotran, the median peak contrast-to-noise ratio (CNR) was 20.7 (25th percentile, 16.3; 75th percentile, 22.6), 24.2 (25th percentile, 19.3; 75th percentile, 28.5), 16.4 (25th percentile, 13.7; 75th percentile, 20.3), and 14.0 (25th percentile, 11.4; 75th percentile, 16.8), respectively. Both doses of VSOP-C184 yielded significantly higher CNR (P < .05) than the other two agents. At T2-weighted turbo spin-echo imaging with 0.015-mmol Fe/kg VSOP-C184, 0.025-mmol Fe/kg VSOP-C184, gadopentetate dimeglumine, and ferucarbotran, the median CNR was 15.0 (25th percentile, 13.4; 75th percentile, 21.3), 15.7 (25th percentile, 14.5; 75th percentile, 19.8), 11.3 (25th percentile, 8.2; 75th percentile, 12.2), and 15.7 (25th percentile, 12.5; 75th percentile, 22.4), respectively. There was no significant difference between VSOP-C184 and ferucarbotran; both had a significantly higher CNR than did gadopentetate dimeglumine. CONCLUSION: VSOP-C184 produces higher liver-to-tumor contrast at dynamic T1-weighted imaging than does gadopentetate dimeglumine; at delayed T2-weighted imaging, the contrast is comparable to that achieved with ferucarbotran.

Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle.

The ability to image cardiomyocyte apoptosis in vivo with high-resolution MRI could facilitate the development of novel cardioprotective therapies. The sensitivity of the novel nanoparticle AnxCLIO-Cy5.5 for cardiomyocyte apoptosis was thus compared in vitro to that of annexin V-FITC and showed a high degree of colocalization. MRI was then performed, following transient coronary artery (LAD) occlusion, in five mice given AnxCLIO-Cy5.5 and in four mice given an identical dose (2 mg Fe/kg) of CLIO-Cy5.5. MR signal intensity and myocardial T2* were evaluated, in vivo, in hypokinetic regions of myocardium in the LAD distribution. Ex vivo fluorescence imaging was performed to confirm the in vivo findings. Myocardial T2* was significantly lower in the mice given AnxCLIO-Cy5.5 (8.1 versus 13.2 ms, P<0.01), and fluorescence target to background ratio was significantly higher (2.1 versus 1.1, P<0.01). This study thus demonstrates the feasibility of obtaining high-resolution MR images of cardiomyocyte apoptosis in vivo with the novel nanoparticle, AnxCLIO-Cy5.5.

Magneto/optical annexin V, a multimodal protein.

Multimodal proteins, or proteins labeled with both fluorescent and magnetic reporter groups, can be used in a wide range of applications including FACS or fluorescence microscopy, MRI and or near-infrared based optical imaging, or to fractionate cells by magnetic cell sorting. A problem with multimodal proteins, however, is the need to maximize bioactivity, often achieved by minimizing the number of modification points of the protein, while attaching fluorescent and magnetic labels. Here we describe the synthesis of a magneto/optical form of annexin V, achieved by reacting the amino-CLIO nanoparticle with Cy5.5 and SPDP, to produce a fluorescent, sulfhydryl reactive nanoparticle. A single reactive sulfhydryl group was added to annexin V by reaction with SATA that preserved the protein's ability to bind apoptotic Jurkat T cells. Reacting SATAylated annexin V with an SPDP activated nanoparticle yielded Anx-CLIO-Cy5.5, a magneto/optical form of annexin V. The binding of Anx-CLIO-Cy5.5 was specific for apoptotic Jurkat T cells and had an EC(50) of 3.66 nM. This was comparable to the strength of the interaction of unmodified annexin V with apoptotic cells, measured as the displacement of FITC-annexin by annexin V (2.4 nM). Our conjugation strategy preserves the strength of the interaction between annexin V and apoptotic cells, while yielding a probe, Anx-CLIO-Cy5.5, that is readily detectable by standard MR imaging or NIRF optical methods.

Visualization of antitumor treatment by means of fluorescence molecular tomography with an annexin V-Cy5.5 conjugate.

In vivo imaging of treatment responses at the molecular level could have a significant impact on the speed of drug discovery and ultimately lead to personalized medicine. Strong interest has been shown in developing quantitative fluorescence-based technologies with good molecular specificity and sensitivity for noninvasive 3D imaging through tissues and whole animals. We show herein that tumor response to chemotherapy can be accurately resolved by fluorescence molecular tomography (FMT) with a phosphatidylserine-sensing fluorescent probe based on modified annexins. We observed at least a 10-fold increase of fluorochrome concentration in cyclophosphamide-sensitive tumors and a 7-fold increase of resistant tumors compared with control studies. FMT is an optical imaging technique developed to overcome limitations of commonly used planar illumination methods and demonstrates higher quantification accuracy validated by histology. It is further shown that a 3-fold variation in background absorption heterogeneity may yield 100% errors in planar imaging but only 20% error in FMT, thus confirming tomographic imaging as a preferred tool for quantitative investigations of fluorescent probes in tissues. Tomographic approaches are found essential for small-animal optical imaging and are potentially well suited for clinical drug development and monitoring.

Optimal modification of annexin V with fluorescent dyes.

The many uses of chemically modified annexin Vs necessitate an understanding of the optimal degree of modification and modification sites of the protein. When reacted with the N-hydroxysuccinimide ester of Cy5.5, annexin V with one modification per mole of protein retained its affinity for phosphatidylserine of apoptotic cells, whereas modification with two dyes per mole of protein caused a complete loss of activity. A tryptic digest LC/MS method was used to identify the modification sites as either of two closely spaced lysine residues, in position 286 or 290. The crystal structure indicated the location of these lysines was distal to the phosphatidylserine binding sites on annexin V. These results can be used to develop active or inactive fluorescent control annexin V proteins and to suggest strategies for attaining higher levels of modification with retention of bioactivity.

Surface-functionalized nanoparticle library yields probes for apoptotic cells.

We have developed techniques for the efficient synthesis and screening of small libraries of surface-functionalized nanoparticles for the recognition of specific types of cells. To illustrate this concept we describe the development of a nanoparticle that preferentially recognizes apoptotic Jurkat cells in a manner similar to the apoptosis-recognizing protein annexin V. The nanoparticle, which is detectable by fluorescence or NMR relaxometry, was analyzed for the ability to recognize normal and apoptotic cells by fluorescence-activated cell sorting (FACS) analysis and fluorescence microscopy. The capability to develop nanoparticles which interact with specific target cells could be applied to the design of materials for diverse applications including quantum dots, which serve as fluorescence tracers, colloidal gold, which serves as a tracer for electron micrographs, or the crystalline forms of drugs.

Optical imaging of apoptosis as a biomarker of tumor response to chemotherapy.

A rapid and accurate assessment of the antitumor efficacy of new therapeutic drugs could speed up drug discovery and improve clinical decision making. Based on the hypothesis that most effective antitumor agents induce apoptosis, we developed a near-infrared fluorescent (NIRF) annexin V to be used for optical sensing of tumor environments. To demonstrate probe specificity, we developed both an active (i.e., apoptosis-recognizing) and an inactive form of annexin V with very similar properties (to account for nonspecific tumor accumulation), and tested the agents in nude mice each bearing a cyclophosphamide (CPA) chemosensitive (LLC) and a chemoresistant LLC (CR-LLC). After injection with active annexin V, the tumor-annexin V ratio (TAR; tumor NIRF/background NIRF) for untreated mice was 1.22+/-0.34 for LLC and 1.43+/-0.53 for CR-LLC (n=4). The LLC of CPA-treated mice had significant elevations of TAR (2.56+/-0.29, P=.001, n=4), but only a moderate increase was obtained for the CR-LLC (TAR=1.89+/-0.19, P=.183). The in vivo measurements correlated well with terminal deoxyribosyl transferase-mediated dUTP nick end labeling indexes. When inactive Cy-annexin V was used, with or without CPA treatment and in both CCL and CR-CCL tumors, tumor NIRF values ranged from 0.91 to 1.17 (i.e., tumor were equal to background). We conclude that active Cy-annexin V and surface reflectance fluorescence imaging provide a nonradioactive, semiquantitative method of determining chemosensitivity in LLC xenografts. The method maybe used to image pharmacologic responses in other animal models and, potentially, may permit the clinical imaging of apoptosis with noninvasive or minimally invasive instrumentation.

Annexin V-CLIO: a nanoparticle for detecting apoptosis by MRI.

Annexin V, which recognizes the phosphatidylserine of apoptotic cells, was conjugated to crosslinked iron oxide (CLIO) nanoparticles, a functionalized superparamagnetic preparation developed for target-specific magnetic resonance imaging (MRI). The resulting nanoparticle had an average of 2.7 annexin V proteins linked per CLIO nanoparticle through disulfide bonds. Using camptothecin to induce apoptosis, a mixture of Jurkat T cells (69% healthy and 31% apoptotic) was incubated with annexin V-CLIO and was applied to magnetic columns. The result was an almost complete removal of the apoptotic cells (> 99%). In a phantom MRI experiment, untreated control cells (12% apoptotic cells, 88% healthy cells) and camptothecin-treated cells (65% apoptotic cells, 35% healthy cells) were incubated with either annexin V-CLIO (1.0, 0.5, and 0.1 microgram Fe/mL) or with unlabeled CLIO. A significant signal decrease of camptothecin-treated cells relative to untreated cells was observed even at the lowest concentration tested. Unmodified CLIO failed to cause a significant signal change of apoptotic cells. Hence, annexin V-CLIO allowed the identification of cell suspensions containing apoptotic cells by MRI even at very low concentrations of magnetic substrate. Conjugation of annexin V to CLIO affords a strategy for the development of a MRI imaging probe for detecting apoptosis.